Enhanced heterologous production of lipoxygenases

ABSTRACT

The invention is directed to the enhanced expression and purification of lipoxygenase enzymes. These enzymes are of wide-spread industrial importance, often produced in heterologous microbial systems. Preferably, the lipoxygenase produced by the methods of the invention is a plant-derived enzyme and expressed at high-levels in a microbial system that includes a protease-deficient host and one or more chaperone expression plasmids. The invention is also directed to amino acid and nucleic acid fragments of the lipoxygenase enzyme including fragments in expression constructs encoding all or a portion of one or more lipoxygenase genes. The invention is also directed to methods of manufacturing bread and other food and also non-food products with lipoxygenase manufactured by the methods of the invention.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional applicationSer. No. 14/263,105, filed Apr. 28, 2014, which claims priority to U.S.Provisional Application No. 61/817,077, filed Apr. 29, 2013, both of thesame title and both of which are incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 28, 2014, isnamed 3060.002.US_SL.txt and is 29,528 bytes in size.

BACKGROUND

1. Field of the Invention

The invention is directed to systems, compositions and methods for theexpression and purification of lipoxygenases, to amino acid and nucleicacid sequences of all or portions of lipoxygenases, to molecularconstructs for the expression of lipoxygenases, and, in particular, tomethods for the large scale production and use of lipoxygenases in foodproducts.

2. Description of the Background

Lipoxygenases (LOXs; EC1.13.11_), also known as lipoxydases, arenon-heme iron-containing dioxygenases distributed in plants and animals.LOXs catalyze hydroperoxidation of polyunsaturated fatty acids in thefirst step of fatty acid metabolite synthesis, to produce an unsaturatedfatty acid hydroperoxide. A LOX definition according to enzymeclassification is linoleate: oxygen oxidoreductase (for plant LOX) andarachidonate: oxygen oxidoreductase (for mammalian LOX). In plants, themost common LOX substrates linoleic acid and linolenic acids areconverted into a variety of bioactive mediators involved in plantdefense, senescence, seed germination, as well as plant growth anddevelopment (Grechkin A. Recent developments in biochemistry of theplant lipoxygenase pathway; Prog Lipid Res. 1998 Nov. 37(5):317-52).Lipoxygenases with different specificities, subcellular location, andtissue-specific expression patterns have been identified as ubiquitouslyfound across kingdoms from bacteria to mammals.

LOXs are of commercial value in various industries including but notlimited to food-related applications in food processing including breadmaking (bleaching and improved texture), aroma and flavor enhancement aswell as for production of perfumes, paint driers (lipoxygenases:potential starting biocatalysts for the synthesis of signalingcompounds. Joo Y C, Oh D K. 2012) and pitch control in softwood pulp(Microbial and enzymatic control of pitch in the pulp and paperindustry, Ana Gutiérrez & José C. del Río & Angel T. Martínez, ApplMicrobiol Biotechnol (2009) 82:1005-1018). Lipoxygenase is present inseeds (e.g. soybeans), grains and many other plant tissues. In thepresence of oxygen, lipoxygenase oxidizes unsaturated fatty acids andproduces lipid hydroperoxides, which improve dough structure through theoxidation of unsaturated fatty acids and subsequently react withspecific chemical components of flour. As a consequence, dough stabilityand rising is increased, which either or together can increase thevolume of the final product.

Regarding the processing of bread, lipoxygenase enzymes offer anadvantage over current chemical additives. The flour ingredient industryhad long been using chemical bleach, mostly benzoyl peroxide (BPO).Because of potential health concerns over BPO, some Euro countries andChina banned the usage of BPO in flour. In the U.S., BPO is still widelyused, but the demand keeps shirking although there is currently no safealternative. Azodiformamide is another chemical alternative, but thedosage is limited to 40 ppm. At this trace dosage, the bleaching effectis quite restrained. In contrast, enzyme additives especially LOXs canreplace chemicals to allow for the processing of flour, resulting in thebleaching of bread and its improved texture. In addition, lipidhydroperoxidases decolorize dough and oxidizes carotinoids, convertingthem into colorless compounds. This blanching of the dough results inlighter colored product, which is highly desired.

With regard to enzymes employed in the food industry, regulationsfrequently require enzymes to be recognized or proven as safe for use.In the case of lipoxygenases, considering that they are ubiquitouslyfound in plants and consumed by humans and animals alike, plantlipoxygenases are considered safe for use, and therefore, of major valueto the industry. Although soy extracts containing high levels oflipoxygenases have been used as an additive for bread manufacturing, soyproduces an undesirable taste and smell and, accordingly, not often auseful option.

Because of plant LOX value, many attempts at high-level expression ofrecombinant plant derived LOX from soy, rice, potato and other sourcesby heterologous expression in microbial hosts including, but not limitedto bacteria such as E. coli (BL21 strain), Bacilli, and in yeast hasbeen attempted, though production was limited [3-8]. The best of these,although still a poor expression from E. coli, was observed at very coldtemperatures [8]. Only one lipoxygenase was produced in Bacilli athigh-levels (˜160 mg/L), but the lipoxygenase was from a bacterialenzyme, not a plant and consequently not approved for use in the humanfood industry [9, 10]. In addition, yields still could not achievedesired levels. Accordingly, a need exists for high level expression ofplant lipoxygenases that is generally recognized as safe for use infoods, and easily produced in large quantities.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs, and provides new methodsand compositions involving the heterologous expression, purification anduse of lipoxygenases.

One embodiment of the invention is directed to the heterologousexpression of lipoxygenases in microbes.

Another embodiment of the invention is directed to methods for thepurification of lipoxygenases, preferably from heterologous expressionsystems according to the invention.

Another embodiment of the invention is directed to lipoxygenasepolypeptide and nucleic acids sequences and molecular constructs oflipoxygenase coding sequences, preferably for the high level expressionof lipoxygenase as compared to expression in wild-type host cells.Preferably wild-type host cells are cells that do not contain a proteasedeficiency and/or cells that do not contain one or more chaperones.

Another embodiment of the invention is directed to methods for themanufacture of bread products comprising adding lipoxygenases of theinvention to a dough containing unsaturated fatty acids and/orcarotinoids. Preferably the lipoxygenase reacts with components of theflour forming lipid hydroperoxides increasing the stability of the doughand enhancing the volume of the baked goods.

Another embodiment of the invention is directed to purified lipoxygenaseenzyme made by the methods of the invention. When the purified enzyme isadded to dough, another embodiment of the invention comprises productsmade with the purified enzyme added to dough such as, preferably, breadproducts. The manufacture of bread products of the invention preferablycomprises adding lipoxygenases to a dough containing unsaturated fattyacids and/or carotinoids. Preferably the lipoxygenase reacts withcomponents of the flour forming lipid hydroperoxides increasing thestability of the dough and enhancing the volume of the baked goods.

Other embodiments and advantages of the invention are set forth in partin the description, which follows, and in part, may be obvious from thisdescription, or may be learned from the practice of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 Western analysis of SLP1 indicates varying profiles ofdegradation. M=marker. Different K12 cells with different genotypes arepresented in sets of “U” (uninduced) and “I” (induced).

FIG. 2 Co-expression of the GroEL-GroES chaperone enhances SLP1production. Four chaperones A-D were co-expressed in E. coli with SLP1.Left Panel: SLP1 is directed detected as a weak band in SDS-PAGE as aresult of Co-expression with Chaperone B. Right Lane: Enhancedexpression of SLP1 with co-expressed GroEL-GroES is confirmed bystandard Western analysis.

FIG. 3 Single step purification of SLP1 from the 424 vector (nativeprotein sequence, no polyhistidine tag). All lanes: SLP1 lacking a histag was eluted from Zinc-NTA (IMAC) columns and run in SDS PAGE followedby protein staining: Lanes 1—crude lysate; Lane 2—column flow-through;Lane 3—column wash; Lanes 4-7—Zinc-NTA (IMAC) elution with SLP1 loadedin the presence of 0, 2, 5 and 10 mM imidazole, each eluted with 80 mMimidazole.

FIG. 4 E. coli cell lines used to verify experiments.

FIG. 5 SLP1 expression in E. coli; SDS PAGE protein gel of whole cellK12 E. coli lysate expressing SLP1.

DESCRIPTION OF THE INVENTION

Lipoxygenase enzymes (also referred to herein as LOX) are widely used incommercial processing of food products, the manufacture of perfumes andpainting products, and in the processing of wood pulp. Although alllipoxygenase catalyze the same basic function, only plant lipoxygenaseshave been approved by the United States Food and Drug Administration foruse in foods and food products. Despite their broad uses, lipoxygenaseenzymes are only expressed at low levels and, consequently, commercialquantities are both expensive and difficult to produce.

Despite previous failures in achieving high level LOX expression, it hasbeen surprisingly discovered that considerable enhancement of plantlipoxygenase expression can be achieved. At least part of thishigh-level of expression is attributed to the selection of sequencesbeing expressed, expression of the sequences in a protease deficienthost, and/or the co-expression with one or more chaperone plasmidsequences. Preferable, the increased expression achieved is at a higherlevel than expression in host cells that do not contain a proteasedeficiency and/or cells that do not contain one or more chaperoneplasmids. Preferably the expression of the one or more proteases iseliminated, reduced to an undetectable level using conventionaldetection or reduced by at least 90%, all as compared to wild-typeexpression levels.

One embodiment of the invention comprises a system containing abacterial cell host, preferably with a deficiency or one or moreproteases, containing a coding sequence for lipoxygenase enzyme andpreferably a chaperone system comprising one or more chaperonemolecules. The system is preferably inducible and also preferablymaintained from about 10° C. to about 37° C. for a period of time formaximal expression of enzyme product. The period of time is preferablyfrom minutes to hours to days, and more preferably from about 1 to about24 hours, more preferably from 2 to 12 hours and more preferably fromabout 2 to about 4 hours. The cells are preferably maintained attemperatures from about 15° C. to about 25° C. during this period.

The lipoxygenase enzyme may be derived from animal or bacterial cells,and is preferably derived from plant cells. Expression constructs maycontain all or a portion of the lipoxygenase gene or coding region.Preferably constructs contain a portion of the coding region sufficientto create functional lipoxygenase activity. Preferably the constructs ofthe invention encode the sequences of SEQ ID NOs 1-3, or contain thenucleic acid sequences of SEQ ID NOs 4-6. Also preferably the sequenceis a functional sequences that generates functional lipoxygenaseactivity.

Preferably the host cell is a microorganism that rapidly andeconomically proliferates in vitro such as, for example, one or more ofthe bacterial cell strains of K12 cells, E. coli cells, Bacillus cells,Lactococci or yeast cells. Also preferably, the host cells contain oneor more protease deficiencies as compared to wild-type cells. For E.coli host cells, the deficiency is preferably of one or more of theproteases Lon, OMPT, and/or Lon/ClpP.

Preferably the host cells further contain one or more chaperone plasmidexpression vectors. Chaperones function in assisting protein folding,benefiting the co-expressed molecules.

Expression of lipoxygenase in the systems of the invention typicallyinvolves inducing expression of the lipoxygenase sequence and alsopreferably the chaperone sequences before, during or after expression ofthe lipoxygenase, and preferably simultaneously or nearly simultaneouslyto allow for maximal expression of the enzyme.

Lipoxygenase produced according to methods of the invention can befurther isolated and purified. Preferably, purification of lipoxygenaseproduced according to the methods of the invention involves contact thewith immobilized-metal affinity chromatography media. The enzyme remainsbound and can be washed with wash buffer and subsequently eluted withelution buffer.

Preferably the increased lipoxygenase expression of the invention is 5fold greater as compared to expression in wild-type cells (e.g., cellsthat are not protease deficient and/or cells without one or moreexpression chaperones), more preferably 10 fold greater, more preferably50 fold greater, more preferably 100 fold greater, more preferably 200fold greater, more preferably 300 fold greater, more preferably 400 foldgreater, and more preferably 500 fold greater or more.

Lipoxygenase made according to the invention is preferably useful in themanufacture of food products such as bread products (for either, or bothbleaching and improving texture), the manufacture of paints thinners,perfumes, aroma and flavor enhancers, as signaling compounds, and forpitch control in softwood pulp in paper industry.

The following examples illustrate embodiments of the invention, butshould not be viewed as limiting the scope of the invention.

EXAMPLES Example 1 LOXs Employed For Protein Production

SLP1 (seed linoleate 13S-lipoxygenase-1 [Glycine max] NCBI ReferenceSequence: NP_001236153.1, length 839 amino acids) and SLP3 seedlinoleate 9S-lipoxygenase-3 [Glycine max] NCBI Reference Sequence:NP_001235383.1) were employed as LOXs for production in microbes. Inaddition, a shortened version of SLP1 (herein minilox) from amino acidSerine 278 containing an additional methionine before the Serine 278were cloned and expressed in microbes.

Example 2 Synthesis of DNA Encoding Protein Sequences for SLP1, SLP3 andMinilox Optimized For Expression

Optimal gene codon usage in plants and bacteria differ. New DNA encodingsequences for SLP1, SLP3 and minilox were determined and syntheticallygenerated according to instructions (Genscript USA Inc.). The sequencefor minilox was identical as that of SLP1 with the exception of havingan ATG encoding for an initiator methionine prior to nucleotide basesencoding for SLP1 Serine 278. Optimized sequences with desired cloningsites were created.

Example 3 Cloning of Soybean Lipoxygenase 1 (SLP1) and 3 (SLP3) andMiniLox

Initially, SLP1 and SLP3 were cloned into the pET 47b vector (Novogen)using SmaI-XhoI restriction sites, so that each contained the pET47binitiating methionine and a 6X histidine tag (SEQ ID NO 7). The SLP1 andminilox encoding DNA were then transferred to the DNA2.0 expressionvector 424 purple, a low copy number plasmid without the histidine tagsusing NdeI-Xhol sites, so that expressed proteins would not contain thehistidine tags. Similarly, the SLP1 encoding sequence was cloned intothe 424-purple vector (herein 424 vector) with the exception of usingNdeI-EcoRI cloning sites. The SLP1 encoding sequences were thentransferred to the DNA2.0 purple-444 vector (herein 444 vector), a highcopy number plasmid using restriction sites Ndel-XhoI. The vectorscontained promoters for expression of the insert DNA with the pET47containing a T7-promotor, and the DNA2.0 vectors contained aT5-promotor.

Example 4 Expression of SLP1, Minilox and SLP3

Vectors were transfected into cell lines. Initially, expression of SLP1was performed with the 6 histidine (SEQ ID NO 7) tagged SLP1 vector inE. coli BL21 cells, an E. coli B cell line suitable for the expressionof the pET47b vectors. Thereafter, all expression was performed in E.coli K12 strains. Expression was tested in LB media, with 50-100 μg/m1ampicillin, and induction of expression for all vectors was with 0.5-1mM IPTG.

Example 5 Activity Assay

The activity assay utilized linoleic acid as a substrate andcolorimetric detection of product. Detected values for the assay varieddepending on the substrate preparation, age of substrate, and substratebatch, which may be subject to variation due to oxidation from theenvironment. As such, approximate expression levels of SLP1 in BL21 arepresented as 1 unit/cell OD550 SLP1-LB culture and relative andapproximate values for expression in other strains is relative to theBL21 expression. Cell OD550 is defined as a cell density at OD550.

Example 6 Improvement of SLP1 Production

SLP1 expressed with or without a histidine tag using the pET47b vectorand BL21 cells was very poor when induced at room temperature. Thestandard level of activity, 1 unit/OD cells was established forinduction at 15° C. with and overnight expression. Dramatically improvedactivity was observed using the purple-424 vector (herein 424 vector),in the K12 HMS 174 cell line (4 units/Cell OD550). Unlike BL21 cells,activity was also observed when induced at 20° C.-25° C. with overnightexpression. Slp1 activity could be further enhanced by growing cells at15° C. for up to several days. In all E. coli strains tested, growth at37C of was found to generate little or no SLP1 activity, and proteindegradation products were observed upon western analysis (FIG. 2).

An additional increase in activity was discovered using proteasedeficient E. coli K12 strains with the 424 vector. Lon, OMPT, orLon/ClpP mutants all showed a further minimum two-fold increase inactivity with (˜10 units/cell OD550). The specific E. coli cell lineswith specific protease deficiencies also showed some similarcharacteristics of protein degradation (FIG. 3) yet some cell lines hadless degradation than others, signifying that proteases play a role inthe limited production of lipoxygenases.

An additional enhancement of activity was observed when using the 444high plasmid copy vector in the K12 Pam155 (lon protease deficient) E.coli cell line and with chaperones. Chaperone plasmid sets consisting offive different plasmids from Takara Bio Inc. each designed to express asingle or multiple molecular chaperone sets can enable optimal proteinexpression and folding and reduce protein misfolding. Each Takaraplasmid carries an origin of replication (ORF) derived from pACYC and achloramphenicol-resistance gene (Cmr) gene, which allows the use of E.coli expression systems containing ColE1-type plasmids that conferampicillin resistance. The chaperone genes are situated downstream ofthe araB or Pzt-1 (tet) promoters and, as a result, expression of targetproteins and chaperones can be individually induced if the target geneis placed under the control of different promoters (e.g., lac). Theseplasmids also contain the necessary regulator (araC or tet^(r)) for eachpromoter. Takara Bio Inc. plasmids containing chaperones or sets thereofeither tetracycline or arabinose inducible were coexpressed with SLP1.These include: groES-groEL, dnaK-dnaJ-grpE, groES-groEL-tig, or tig inplasmids (TakaraBo Inc.). Expression of SLP1 in the presence ofgroES-groEL alone or with tig (groES-groEL-tig) enhanced the amount ofactive enzyme produced roughly to 40-60 units/cell OD. Activity wasoptimal at 15° C. but also observed at or below 25° C. At 37° C.,expression was more limited.

Expression of SLP1 in the Pam153 cell with concomitant GroESL chaperoneexpression, in LB, produces 68 micrograms of SLP1 per milliliter at abacterial OD550 of 3, when grown in test tubes at 37° C. and induced at20° C. overnight. However, SLP1 expression in an E. coli strain that isnot a protease deficient strain and without chaperone expression caneither not be detected at all with standard SDS PAGE analysis, orwestern analysis, or expresses less than 1 μg per milliliter LB undersimilar conditions at an OD550 of 3 (see FIG. 4). In general, expressionof SLP1 in E. coli strains grown and induced under optimal conditionswas undetectable or less than 1 microgram per milliliter whenappropriate chaperones were absent and strains were not proteasedeficient. However when expressing SLP1 in E. coli K12 proteasedeficient strains with co-expression of an appropriate chaperone, 68micrograms of SLP1 per milliliter at a bacterial OD550 of 3 wasattained.

Example 7 Purification of SPL1

Purification of SLP1 with the 6Xhis tag (SEQ ID NO 7) was highlyeffective using standard Ni-NTA IMAC purification. In the 424 or 444vectors lacking the 6Xhis tag (SEQ ID NO 7), where SLP1 was encoded bythe native SLP1 sequence alone, IMAC was equally efficient though undermodified conditions. Nickel and zinc were each tested with similarresults and calcium or other divalent metals should do as well. Buffersfor IMAC were either 50 mM phosphate or Tris-HCl at pH 7-9, with 400 mMNaCl and 10% glycerol. Cells were disrupted using B-PER (Peirce) or by ahomogenizer, in the presence of PMSF as a protease inhibitor. EmployingZinc-NTA, it was discovered that loading the sample in buffer with 10 mMimidazole and elution in buffer with 80 mM imidazole was effective inpurification of SLP1. Other column media that effectively binds SLP1include MonoQ and DEAE, but not negatively charged resins.

Example 8 Novel Information Provides Improved SLP1 Expression

Preliminary studies indicate that relatively poor production of SLP1 isthe result of rapid proteolysis accompanied by improper folding of theenzyme. The limited soluble SLP1 and lack of insoluble protein suggeststhat most of the protein produced was rapidly degraded. Degradationproducts of SLP1 are visible in different E. coli strains with differentprotease deficient genetic backgrounds (see FIG. 1). An increase in bothactive enzyme and total protein was observed when inducing at suboptimalgrowth temperatures, where proteases are less functional. A relativeincrease in production and activity of SLP1 when protein folding isenhanced by an over-expressed chaperone.

Example 9 High Level Expression of Lipoxygenase in the E. Coli, K12,

Unless otherwise stated, all bacterial media employed in this examplewas Luria Broth (herein LB, consisting of 10 grams Tryptone, 5 gramsYeast Extract, and 10 grams NaCl, dissolved in 1 liter water, andsterilized for a minimum of 20 minutes in an autoclave). SoybeanLipoxygenase 1 (herein SLP1) was expressed from a plasmid transfectedinto E. Coli K12 cells. FIG. 5 represents an SDS-PAGE protein gel ofwhole cell soluble proteins extracted from the K12 cells employing thecommercial B-PER Protein Extraction Reagent (Pierce, Cat#78243),following company protocols. The highest level of soluble SLP1 proteinrelative to total soluble protein in the cell extract was 30% or greaterand approximated at 34% as estimated by the ImageJ (National Instituteof Health public software) analysis software. These levels areconsistent with high level production of the enzyme. M=marker, 1Uninduced, 2 SLP1 induced with 0.5 mM IPTG 3&4 Induced with 0.5 mM IPTGand expressing a molecular chaperone.

CITED REFERENCES

-   1. Permiakova, M. D. and V. A. Trufanov, Effect of soybean    lipoxygenae on baking properties of wheat flour, Prikl Biokhim    Mikrobiol, 2011. 47(3): p. 348-54.-   2. Permiakova, M. D., et al., [Role of lipoxygenase in the    determination of wheat grain quality]. Prikl Biokhim    Mikrobiol, 2010. 46(1): p. 96-102.-   3. Kanamoto, H., M. Takemura, and K. Ohyama, Cloning and expression    of three lipoxygenase genes from liverwort, Marchantia polymorpha    L., in Escherichia coli. Phytochemistry, 2012. 77: p. 70-8.-   4. Osipova, E. V., et al., Recombinant maize 9-lipoxygenase:    expression, purification, and properties. Biochemistry Biokhimi    , 2010. 75(7): p. 861-5.-   5. Hwang, I. S. and B. K. Hwang, The pepper 9-lipoxygenase gene    CaLOX1 functions in defense and cell death responses to microbial    pathogens. Plant Physiol, 2010. 152(2): p. 948-67.-   6. Padilla, M. N., et al., Functional characterization of two    13-lipoxygenase genes from olive fruit in relation to the    biosynthesis of volatile compounds of virgin olive oil. J Agric Food    Chem, 2009. 57(19): p. 9097-107.-   7. Knust, B. and D. von Wettstein, Expression and secretion of    pea-seed lipoxygenase isoenzymes in Saccharomyces cerevisiae. Appl    Microbiol Biotechnol, 1992. 37(3): p. 342-51.-   8. Steczko, J., et al., Effect of ethanol and low-temperature    culture on expression of soybean lipoxygenase L-1 in Escherichia    coli. Protein Expr Purif, 1991. 2(2-3): p. 221-7.

Sequence Information

SLP1 polypeptide sequence (SEQ ID NO 1)MFSAGHKIKGTVVLMPKNELEVNPDGSAVDNLNAFLGRSVSLQLISATKADAHGKGKVGKDTFLEGINTSLPTLGAGESAFNIHFEWDGSMGIPGAFYIKNYMQVEFFLKSLTLEAISNQGTIRFVCNSWVYNTKLYKSVRIFFANHTYVPSETPAPLVSYREEELKSLRGNGTGERKEYDRIYDYDVYNDLGNPDKSEKLARPVLGGSSTFPYPRRGRTGRGPTVTDPNTEKQGEVFYVPRDENLGHLKSKDALEIGTKSLSQIVQPAFESAFDLKSTPIEFHSFQDVHDLYEGGIKLPRDVISTIIPLPVIKELYRTDGQHILKFPQPHVVQVSQSAWMTDEEFAREMIAGVNPCVIRGLEEFPPKSNLDPAIYGDQSSKITADSLDLDGYTMDEALGSRRLFMLDYHDIFMPYVRQINQLNSAKTYATRTILFLREDGTLKPVAIELSLPHSAGDLSAAVSQVVLPAKEGVESTIWLLAKAYVIVNDSCYHQLMSHWLNTHAAMEPFVIATHRHLSVLHPIYKLLTPHYRNNMNINALARQSLINANGIIETTFLPSKYSVEMSSAVYKNWVFTDQALPADLIKRGVAIKDPSTPHGVRLLIEDYPYAADGLEIWAAIKTWVQEYVPLYYARDDDVKNDSELQHWWKEAVEKGHGDLKDKPWWPKLQTLEDLVEVCLIIIWIASALHAAVNFGQYPYGGLIMNRPTASRRLLPEKGTPEYEEMINNHEKAYLRTITSKLPTLISLSVIEILSTHASDEVYLGQRDNPHWTSDSKALQAFQKFGNKLKEIEEKLVRRNNDPSLQGNRLGPVQLP YTLLYPSSEEGLTFRGIPNSISISLP3 polypeptide sequence (SEQ ID NO 2)MLGGLLHRGHKIKGTVVLMRKNVLDVNSVTSVGGIIGQGLDLVGSTLDTLTAFLGRSVSLQLISATKADANGKGKLGKATFLEGIITSLPTLGAGQSAFKINFEWDDGSGIPGAFYIKNFMQTEFFLVSLTLEDIPNHGSIHFVCNSWIYNAKLFKSDRIFFANQTYLPSETPAPLVKYREEELHNLRGDGTGERKEWERIYDYDVYNDLGDPDKGENHARPVLGGNDTFPYPRRGRTGRKPTRKDPNSESRSNDVYLPRDEAFGHLKSSDFLTYGLKSVSQNVLPLLQSAFDLNFTPREFDSFDEVHGLYSGGIKLPTDIISKISPLPVLKEIFRTDGEQALKFPPPKVIQVSKSAWMTDEEFAREMLAGVNPNLIRCLKDFPPRSKLDSQVYGDHTSQITKEHLEPNLEGLTVDEAIQNKRLFLLDHHDPIMPYLRRINATSTKAYATRTILFLKNDGTLRPLAIELSLPHPQGDQSGAFSQVFLPADEGVESSIWLLAKAYVVVNDSCYHQLVSHWLNTHAVVEPFIIATNRHLSVVHPIYKLLHPHYRDTMNINGLARLSLVNDGGVIEQTFLWGRYSVEMSAVVYKDWVFTDQALPADLIKRGMAIEDPSCPHGIRLVIEDYPYTVDGLEIWDAIKTWVHEYVFLYYKSDDTLREDPELQACWKELVEVGHGDKKNEPWWPKMQTREELVEACAIIIWTASALHAAVNFGQYPYGGLILNRPTLSRRFMPEKGSAEYEELRKNPQKAYLKTITPKFQTLIDLSVIEILSRHASDEVYLGERDNPNWTSDTRALEAFKRFGNKLAQIENKLSERNNDEKLRNRCGPVQMPYTLLLPSSKEGLTFRGIPNSISI Minilox1 polypeptide sequence(SEQ ID NO 3) MSTPIEFHSFQDVHDLYEGGIKLPRDVISTIIPLPVIKELYRTDGQHILKFPQPHVVQVSQSAWMTDEEFAREMIAGVNPCVIRGLEEFPPKSNLDPAIYGDQSSKITADSLDLDGYTMDEALGSRRLFMLDYHDIFMPYVRQINQLNSAKTYATRTILFLREDGTLKPVAIELSLPHSAGDLSAAVSQVVLPAKEGVESTIWLLAKAYVIVNDSCYHQLMSHWLNTHAAMEPFVIATHRHLSVLHPIYKLLTPHYRNNMNINALARQSLINANGIIETTFLPSKYSVEMSSAVYKNWVFTDQALPADLIKRGVAIKDPSTPHGVRLLIEDYPYAADGLEIWAAIKTWVQEYVPLYYARDDDVKNDSELQHWWKEAVEKGHGDLKDKPWWPKLQTLEDLVEVCLIIIWIASALHAAVNFGQYPYGGLIMNRPTASRRLLPEKGTPEYEEMINNHEKAYLRTITSKLPTLISLSVIEILSTHASDEVYLGQRDNPHWTSDSKALQAFQKFGNKLKEIEEKLVRRNNDPSLQGNRLGPVQLPYTLLYPSSEEGLTFRGIPNSISISLP1 DNA optimized encoding sequence (with restriction sites 5′SmaI and 3′ XhoI with stopcodon for cloning into pET47b with 6X histidine tag (SEQ ID NO 7))(SEQ ID NO 4) CCCGGGATGTTTAGTGCTGGTCACAAAATCAAAGGTACCGTGGTCCTGATGCCGAAAAATGAACTGGAAGTCAACCCGGATGGTAGCGCCGTTGATAACCTGAATGCGTTCCTGGGTCGTAGCGTGTCTCTGCAGCTGATTTCCGCCACCAAAGCAGACGCTCACGGCAAGGGTAAAGTTGGCAAAGATACGTTTCTGGAAGGTATTAATACCTCCCTGCCGACCCTGGGTGCCGGTGAATCAGCTTTCAACATCCATTTCGAATGGGATGGTTCAATGGGCATTCCGGGCGCCTTCTACATCAAAAACTACATGCAGGTGGAATTTTTCCTGAAAAGTCTGACCCTGGAAGCAATCTCCAATCAGGGTACGATTCGTTTTGTCTGCAACTCGTGGGTGTATAATACCAAACTGTACAAAAGCGTTCGCATCTTTTTCGCGAACCACACCTATGTTCCGAGCGAAACCCCGGCACCGCTGGTTTCTTACCGTGAAGAAGAACTGAAAAGTCTGCGCGGCAATGGTACCGGCGAACGTAAAGAATATGATCGCATTTATGACTACGATGTTTACAACGACCTGGGCAATCCGGATAAAAGCGAAAAACTGGCCCGTCCGGTCCTGGGCGGTAGCTCTACCTTCCCGTATCCGCGTCGCGGTCGTACCGGTCGTGGTCCGACCGTGACCGATCCGAACACCGAAAAACAGGGCGAAGTCTTTTATGTGCCGCGCGACGAAAATCTGGGCCATCTGAAATCTAAAGATGCCCTGGAAATCGGTACCAAAAGTCTGTCCCAGATTGTGCAACCGGCGTTTGAAAGCGCCTTCGATCTGAAATCTACGCCGATTGAATTTCACTCCTTCCAGGACGTTCATGATCTGTATGAAGGCGGTATCAAACTGCCGCGTGACGTCATTTCAACCATTATCCCGCTGCCGGTGATCAAAGAACTGTACCGCACGGATGGTCAGCACATTCTGAAATTTCCGCAACCGCATGTGGTTCAGGTTTCACAATCGGCGTGGATGACCGATGAAGAATTCGCGCGTGAAATGATCGCCGGCGTTAACCCGTGCGTCATTCGCGGTCTGGAAGAATTTCCGCCGAAAAGCAATCTGGACCCGGCAATCTATGGCGATCAGAGTTCCAAAATTACCGCTGACTCTCTGGACCTGGATGGCTACACGATGGATGAAGCCCTGGGTAGTCGTCGCCTGTTTATGCTGGACTATCACGATATCTTCATGCCGTACGTGCGTCAGATTAACCAACTGAATTCTGCAAAAACCTATGCTACCCGTACGATCCTGTTTCTGCGCGAAGACGGCACGCTGAAACCGGTTGCAATTGAACTGAGCCTGCCGCATTCTGCTGGTGATCTGAGTGCCGCGGTGTCCCAGGTTGTGCTGCCGGCAAAAGAAGGCGTTGAAAGTACCATCTGGCTGCTGGCGAAAGCCTATGTTATTGTCAACGATTCATGTTACCATCAACTGATGTCGCACTGGCTGAATACCCATGCAGCTATGGAACCGTTTGTTATCGCAACGCATCGCCACCTGTCTGTCCTGCACCCGATTTATAAACTGCTGACCCCGCATTACCGTAACAATATGAACATCAATGCACTGGCTCGCCAGAGTCTGATTAACGCGAATGGTATTATCGAAACCACGTTCCTGCCGTCAAAATATTCGGTGGAAATGTCATCGGCCGTTTACAAAAACTGGGTCTTTACCGACCAGGCACTGCCGGCTGATCTGATCAAACGTGGCGTCGCGATTAAAGATCCGAGCACCCCGCATGGTGTGCGTCTGCTGATTGAAGACTATCCGTACGCGGCCGATGGCCTGGAAATCTGGGCAGCTATTAAAACCTGGGTGCAGGAATATGTTCCGCTGTATTACGCACGCGATGACGATGTGAAAAATGACTCCGAACTGCAACACTGGTGGAAAGAAGCTGTTGAAAAAGGTCATGGCGACCTGAAAGATAAACCGTGGTGGCCGAAACTGCAGACCCTGGAAGATCTGGTGGAAGTTTGTCTGATTATCATTTGGATTGCCAGCGCACTGCATGCCGCGGTGAACTTTGGTCAATATCCGTACGGCGGTCTGATTATGAATCGTCCGACCGCAAGCCGTCGCCTGCTGCCGGAAAAAGGCACGCCGGAATACGAAGAAATGATCAACAACCATGAAAAAGCGTACCTGCGCACCATCACGAGCAAACTGCCGACCCTGATTAGCCTGTCTGTTATCGAAATTCTGTCAACGCACGCGTCGGATGAAGTCTATCTGGGTCAGCGTGACAACCCGCATTGGACCAGTGATTCCAAAGCGCTGCAGGCCTTCCAAAAATTCGGCAACAAACTGAAAGAAATCGAAGAAAAACTGGTCCGTCGCAACAATGATCCGAGCCTGCAGGGTAACCGTCTGGGTCCGGTGCAACTGCCGTATACCCTGCTGTATCCGTCCAGTGAAGAAGGTCTGACGTTTCGTGGTATTCCGAACTCCATTTCCATCTGACTCGAGSLP3 DNA optimized encoding sequence (with restriction sites 5′NdeI and 3′ EcoRI and 3′ stop codon for cloning into the pJex purple 424vector from DNA2.0 Inc. (SEQ ID NO 5)CATATGCTGGGCGGCCTGCTGCACCGTGGTCATAAAATCAAGGGCACCGTGGTCCTGATGCGTAAGAACGTCCTGGATGTGAATAGCGTGACCTCGGTCGGCGGTATTATCGGCCAGGGTCTGGACCTGGTGGGTAGCACGCTGGATACCCTGACGGCCTTTCTGGGCCGCTCAGTGTCGCTGCAACTGATCAGCGCAACCAAAGCAGATGCTAACGGCAAAGGCAAGCTGGGCAAGGCGACGTTCCTGGAAGGCATTATCACCTCCCTGCCGACGCTGGGTGCAGGCCAGTCAGCCTTTAAAATTAATTTCGAATGGGATGACGGCTCTGGTATTCCGGGCGCCTTCTACATCAAGAACTTCATGCAGACCGAATTTTTCCTGGTCAGCCTGACGCTGGAAGATATCCCGAATCATGGCTCGATTCACTTTGTGTGCAACAGCTGGATCTACAATGCGAAACTGTTCAAGTCCGATCGCATTTTCTTTGCCAATCAGACCTATCTGCCGTCAGAAACGCCGGCACCGCTGGTTAAATACCGTGAAGAAGAACTGCACAACCTGCGTGGTGACGGTACCGGTGAACGTAAAGAATGGGAACGCATCTACGATTACGACGTTTACAACGATCTGGGTGATCCGGACAAAGGCGAAAACCATGCGCGTCCGGTCCTGGGCGGTAATGACACCTTTCCGTACCCGCGTCGCGGTCGTACCGGTCGTAAACCGACGCGTAAGGATCCGAACAGCGAATCTCGCAGTAATGATGTGTATCTGCCGCGTGACGAAGCCTTTGGTCACCTGAAAAGCTCTGATTTCCTGACGTACGGCCTGAAGTCCGTTTCACAGAACGTCCTGCCGCTGCTGCAAAGCGCATTTGATCTGAATTTCACCCCGCGCGAATTTGATTCGTTCGACGAAGTTCATGGTCTGTATAGCGGCGGTATTAAGCTGCCGACCGACATTATCTCTAAAATCAGTCCGCTGCCGGTGCTGAAGGAAATTTTTCGCACGGATGGCGAACAGGCTCTGAAGTTCCCGCCGCCGAAAGTCATCCAAGTGTCGAAAAGCGCGTGGATGACCGATGAAGAATTTGCACGTGAAATGCTGGCTGGTGTTAACCCGAATCTGATTCGCTGTCTGAAGGATTTCCCGCCGCGTTCCAAACTGGATTCACAGGTGTATGGTGACCACACCAGTCAAATCACGAAAGAACATCTGGAACCGAACCTGGAAGGCCTGACCGTTGATGAAGCTATTCAGAATAAACGTCTGTTTCTGCTGGATCATCACGACCCGATCATGCCGTATCTGCGTCGCATTAATGCGACCTCGACGAAAGCGTACGCCACCCGCACGATCCTGTTCCTGAAGAACGATGGTACCCTGCGTCCGCTGGCCATTGAACTGAGCCTGCCGCATCCGCAGGGTGACCAATCGGGTGCGTTTAGCCAGGTTTTCCTGCCGGCCGATGAAGGCGTCGAAAGTTCCATCTGGCTGCTGGCAAAAGCTTATGTGGTTGTCAACGATTCTTGCTACCATCAGCTGGTGTCTCACTGGCTGAATACCCATGCAGTGGTTGAACCGTTTATTATCGCTACGAACCGCCACCTGTCTGTCGTGCATCCGATCTATAAACTGCTGCATCCGCACTACCGCGACACCATGAACATTAATGGTCTGGCGCGTCTGAGTCTGGTCAACGATGGCGGTGTGATTGAACAGACGTTTCTGTGGGGCCGTTATTCTGTTGAAATGAGTGCCGTTGTCTACAAAGATTGGGTCTTCACCGACCAAGCACTGCCGGCAGACCTGATCAAGCGTGGTATGGCAATTGAAGATCCGTCCTGTCCGCACGGCATCCGTCTGGTGATTGAAGATTATCCGTACACCGTTGACGGTCTGGAAATCTGGGATGCAATTAAAACGTGGGTGCATGAATACGTTTTTCTGTACTACAAGTCTGATGACACCCTGCGCGAAGACCCGGAACTGCAGGCGTGCTGGAAAGAACTGGTGGAAGTTGGTCACGGCGATAAAAAGAACGAACCGTGGTGGCCGAAAATGCAAACCCGTGAAGAACTGGTTGAAGCGTGTGCCATTATCATTTGGACGGCAAGCGCTCTGCATGCGGCCGTGAACTTTGGCCAGTATCCGTACGGCGGTCTGATTCTGAATCGCCCGACCCTGTCTCGTCGCTTCATGCCGGAAAAAGGCAGTGCTGAATATGAAGAACTGCGTAAAAATCCGCAGAAGGCGTACCTGAAAACCATCACGCCGAAATTTCAAACCCTGATTGACCTGAGCGTGATCGAAATTCTGTCCCGCCATGCGTCAGATGAAGTTTATCTGGGTGAACGTGACAACCCGAATTGGACCTCCGATACGCGTGCACTGGAAGCTTTTAAGCGCTTCGGCAACAAACTGGCCCAGATCGAAAACAAGCTGTCAGAACGTAACAACGATGAAAAGCTGCGTAATCGCTGCGGCCCGGTGCAAATGCCGTATACCCTGCTGCTGCCGTCCTCAAAAGAAGGTCTGACGTTCCGTGGTATCCCGAATAGCATTAGCATCTAAGAATTCMinilox optimized encoding sequence (with 5′ NdeI and 3′XhoI restriction sites and 3′ stopcodon for cloning into pJexpress purple 424 vector from DNA2.0 Inc.)(SEQ ID NO 6) CATATGTCTACGCCGATTGAATTTCACTCCTTCCAGGACGTTCATGATCTGTATGAAGGCGGTATCAAACTGCCGCGTGACGTCATTTCAACCATTATCCCGCTGCCGGTGATCAAAGAACTGTACCGCACGGATGGTCAGCACATTCTGAAATTTCCGCAACCGCATGTGGTTCAGGTTTCACAATCGGCGTGGATGACCGATGAAGAATTCGCGCGTGAAATGATCGCCGGCGTTAACCCGTGCGTCATTCGCGGTCTGGAAGAATTTCCGCCGAAAAGCAATCTGGACCCGGCAATCTATGGCGATCAGAGTTCCAAAATTACCGCTGACTCTCTGGACCTGGATGGCTACACGATGGATGAAGCCCTGGGTAGTCGTCGCCTGTTTATGCTGGACTATCACGATATCTTCATGCCGTACGTGCGTCAGATTAACCAACTGAATTCTGCAAAAACCTATGCTACCCGTACGATCCTGTTTCTGCGCGAAGACGGCACGCTGAAACCGGTTGCAATTGAACTGAGCCTGCCGCATTCTGCTGGTGATCTGAGTGCCGCGGTGTCCCAGGTTGTGCTGCCGGCAAAAGAAGGCGTTGAAAGTACCATCTGGCTGCTGGCGAAAGCCTATGTTATTGTCAACGATTCATGTTACCATCAACTGATGTCGCACTGGCTGAATACCCATGCAGCTATGGAACCGTTTGTTATCGCAACGCATCGCCACCTGTCTGTCCTGCACCCGATTTATAAACTGCTGACCCCGCATTACCGTAACAATATGAACATCAATGCACTGGCTCGCCAGAGTCTGATTAACGCGAATGGTATTATCGAAACCACGTTCCTGCCGTCAAAATATTCGGTGGAAATGTCATCGGCCGTTTACAAAAACTGGGTCTTTACCGACCAGGCACTGCCGGCTGATCTGATCAAACGTGGCGTCGCGATTAAAGATCCGAGCACCCCGCATGGTGTGCGTCTGCTGATTGAAGACTATCCGTACGCGGCCGATGGCCTGGAAATCTGGGCAGCTATTAAAACCTGGGTGCAGGAATATGTTCCGCTGTATTACGCACGCGATGACGATGTGAAAAATGACTCCGAACTGCAACACTGGTGGAAAGAAGCTGTTGAAAAAGGTCATGGCGACCTGAAAGATAAACCGTGGTGGCCGAAACTGCAGACCCTGGAAGATCTGGTGGAAGTTTGTCTGATTATCATTTGGATTGCCAGCGCACTGCATGCCGCGGTGAACTTTGGTCAATATCCGTACGGCGGTCTGATTATGAATCGTCCGACCGCAAGCCGTCGCCTGCTGCCGGAAAAAGGCACGCCGGAATACGAAGAAATGATCAACAACCATGAAAAAGCGTACCTGCGCACCATCACGAGCAAACTGCCGACCCTGATTAGCCTGTCTGTTATCGAAATTCTGTCAACGCACGCGTCGGATGAAGTCTATCTGGGTCAGCGTGACAACCCGCATTGGACCAGTGATTCCAAAGCGCTGCAGGCCTTCCAAAAATTCGGCAACAAACTGAAAGAAATCGAAGAAAAACTGGTCCGTCGCAACAATGATCCGAGCCTGCAGGGTAACCGTCTGGGTCCGGTGCAACTGCCGTATACCCTGCTGTATCCGTCCAGTGAAGAAGGTCTGACGTTTCGTGGTATTCCGAAC TCCATTTCCATCTGACTCGAG

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all publications, U.S. and foreign patents and patentapplications, are specifically and entirely incorporated by reference.The term comprising, where ever used, is intended to include the termsconsisting and consisting essentially of. Furthermore, the termscomprising, including, and containing are not intended to be limiting.It is intended that the specification and examples be consideredexemplary only with the true scope and spirit of the invention indicatedby the following claims.

1. A method of producing lipoxygenase enzyme comprising: providing anucleic acid expression construct within a host microorganism, whereinthe construct encodes a plant-derived lipoxygenase enzyme; providing oneor more chaperone plasmids within the host microorganism; and inducingexpression of a lipoxygenase polypeptide encoded by the construct. 2.The method of claim 1, further comprising purifying the expressedlipoxygenase polypeptide and collecting isolated lipoxygenasepolypeptide.
 3. The method of claim 1, wherein the nucleic acidexpression construct encodes all or a functional portion of the sequenceof SEQ ID NO 1, SEQ ID NO 2, or SEQ ID NO
 3. 4. The method of claim 1,wherein the nucleic acid expression construct contains all or afunctional portion of the sequence of SEQ ID NO 4, SEQ ID NO 5, or SEQID NO
 6. 5. The method of claim 1, wherein the host microorganism is abacterial cell containing one or more protease deficiencies.
 6. Themethod of claim 5, wherein the bacterial cell is a strain of K12 cells,E. coli cells, Bacillus cells, Lactoccocus or yeast cells.
 7. The methodof claim 5, wherein the bacterial cell is an organism generallyrecognized as safe for the production of food enzymes.
 8. The method ofclaim 1, wherein the one or more chaperone plasmids are simultaneouslyco-expressed with the lipoxygenase polypeptide.
 9. The method of claim1, wherein inducing comprises maintaining the heterologous microorganismat from 10-37° C. for a period of time.
 10. The method of claim 9,wherein inducing comprises maintaining the heterologous microorganism atfrom 25-35° C. for a period of time.
 11. The method of claim 9, whereininducing comprises maintaining the heterologous microorganism at from10-25° C. for a period of time.
 12. The method of claim 9, whereininducing comprises maintaining the heterologous microorganism at from20-25° C. for a period of time.
 13. The method of claim 9, wherein theperiod of time is from 1 hour to 2 days.
 14. The method of claim 1,wherein purification comprises contacting the expressed lipoxygenase toimmobilized-metal affinity chromatography media.
 15. The method of claim1, wherein the expressed lipoxygenase polypeptide does not contain ahistidine tag.
 16. The method of claim 1, wherein the collectedlipoxygenase polypeptide comprises a plant lipoxygenase.
 17. The methodof claim 1, wherein the expressed lipoxygenase polypeptide does notcontain a histidine tag.
 18. The method of claim 1, wherein solublelipoxygenase polypeptide relative to total soluble protein in the cellextract is 30% or greater.
 19. A composition comprising lipoxygenasepolypeptide made by the method of claim
 1. 20. A method for themanufacture of a bread product, comprising adding the composition ofclaim 19 to a dough.
 21. The method of claim 20, wherein the doughcontains unsaturated fatty acids, carotinoids or both unsaturated fattyacids and carotinoids.
 22. A bread product made by the method of claim21.
 23. A method of producing lipoxygenase enzyme comprising: providinga nucleic acid expression construct within a host microorganism, whereinthe construct encodes a plant-derived lipoxygenase enzyme and the hostmicroorganism is generally recognized as safe for the production of foodenzymes; providing one or more chaperone plasmids within the hostmicroorganism wherein the one or more chaperone plasmids aresimultaneously co-expressed with the lipoxygenase polypeptide; inducingexpression of a lipoxygenase polypeptide encoded by the construct;purifying the expressed lipoxygenase polypeptide by contacting theexpressed lipoxygenase to immobilized-metal affinity chromatographymedia, wherein soluble lipoxygenase polypeptide relative to totalsoluble protein in the cell extract is 30% or greater; and collectingisolated lipoxygenase polypeptide.
 24. An artificial and functionallipoxygenase enzyme produced by the method of claim 23.